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RDT- Enzymes

Once pure samples of DNA have been prepared. This enzyme act on DNA molecules so that it can be shortened. and the control of gene expression. copied into RNA or into new DNA molecules. for studies of DNA biochemistry. To produce this recombinant molecule. the vector.For this purpose special types of Enzymes are required. and modified by the addition or removal of specific chemical groups. as well as the DNA to be cloned. . This has importance in for gene cloning. gene structure. the next step in a gene cloning experiment is construction of the recombinant DNA molecule. lengthened. must be cut at specific points and then joined together in a controlled manner.

or degrade nucleic acid molecules. shorten.The range of DNA manipulative enzymes DNA manipulative enzymes can be grouped into four broad classes. •Ligases join nucleic acid molecules together. •Modifying enzymes remove or add chemical groups. . depending on the type of reaction that they catalyze •Nucleases are enzymes that cut. •Polymerases make copies of molecules.

leaving singlestranded DNA as the product . Distinction between different exonucleases lies in the number of strands that are degraded when a double-stranded molecule is attacked.Nucleases Nucleases degrade DNA molecules by breaking the phosphodiester bonds l Exonucleases remove nucleotides one at a time from the end of a DNA molecule. The greater the length of time that Bal31 is allowed to act on a group of DNA molecules. coli exonuclease III degrade just one strand of a double-stranded molecule. E. the shorter the resulting DNA fragments will be. l Endonucleases are able to break internal phosphodiester bonds within a DNA molecule. The enzyme called Bal31 (purified from the bacterium Alteromonas espejiana) is an example of an exonuclease that removes nucleotides from both strands of a doublestranded molecule .

. DNase I is non-specific in that it attacks DNA at any internal phosphodiester bond. so the end result of prolonged DNase I action is a mixture of mononucleotides and very short oligonucleotides. which is prepared from cow pancreas.S1 endonuclease (from the fungus Aspergillus oryzae) only cleaves single strands deoxyribonuclease I (DNase I). cuts both single and double-stranded molecules .

for example. DNA replication. DNA ligases from most organisms can also join together two individual fragments of doublestranded DNA .Ligases In the cell the function of DNA ligase is to repair single-stranded breaks (“discontinuities”) that arise in double-stranded DNA molecules during.

degrading the existing strand as it proceeds. which is usually prepared from E. and then synthesizes a completely new strand.Polymerases DNA polymerases are enzymes that synthesize a new strand of DNA complementary to an existing DNA or RNA template . Most polymerases can function only if the template possesses a double-stranded region that acts as a primer for initiation of polymerization. coli. DNA polymerase I. This enzyme attaches to a short single-stranded region (or nick) in a mainly double-stranded DNA molecule. .

DNA polymerase I is therefore an example of an enzyme with a dual activity—DNA polymerization and DNA degradation. The special feature of Taq DNA polymerase that makes it suitable for PCR. The Taq DNA polymerase used in the polymerase chain reaction (PCR) is the DNA polymerase I enzyme of the bacterium Thermus aquaticus. Removal of nuclease segment of DNA Polymerase leaves a modified enzyme that retains the polymerase function. . This organism lives in hot springs. meaning that they are resistant to denaturation by heat treatment. The major application of these polymerases is in DNA sequencing. can still synthesize a complementary DNA strandmon a single-stranded template . This modified enzyme. called the Klenow fragment. including the Taq DNA polymerase. are thermostable. and many of its enzymes.

The ability of this enzyme to synthesize a DNA strand complementary to an RNA template is central to the technique called complementary DNA (cDNA) cloning .The final type of DNA polymerase that is important in genetic engineering is reverse transcriptase an enzyme involved in the replication of several kinds of virus. Reverse transcriptase is unique in that it uses as a template not DNA but RNA .

adding phosphate groups onto free 5′ termini. coli. calf intestinal tissue) which removes the phosphate group present at the 5′ terminus of a DNA molecule l Polynucleotide kinase (from E.DNA modifying enzymes enzymes that modify DNA molecules by addition or removal of specific chemical groups. The most important are as follows: l Alkaline phosphatase (from E. coli infected with T4 phage). which has the reverse effect to alkaline phosphatase. .

which adds one or more deoxyribonucleotides onto the 3′ terminus of a DNA molecule .l Terminal deoxynucleotidyl transferase (from calf thymus tissue).

when it was shown that some strains of bacteria are immune to bacteriophage infection. species of bacteria: over 2500 different ones have been isolated and more than 300 are available for use in the laboratory. perhaps all. The bacterium’s own DNA. Restriction occurs because the bacterium produces an enzyme that degrades the phage DNA before it has time to replicate and direct synthesis of new phage particles . the destruction of which would of course be lethal. Each vector molecule must be cleaved at a single position. a phenomenon referred to as host-controlled restriction. The discovery and function of restriction endonucleases The initial observation that led to the eventual discovery of restriction endonucleases was made in the early 1950s.Enzymes for cutting DNA—restriction endonucleases Gene cloning requires that DNA molecules be cut in a very precise and reproducible fashion. is protected from attack because it carries additional methyl groups that block the degradative enzyme action These degradative enzymes are called restriction endonucleases and are synthesized by many. It is important that a very special type of nuclease is needed to carry out this manipulation. to open up the circle so that new DNA can be inserted. .

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Types I and III are rather complex and have only a limited role in genetic engineering. There are RE cut at four. HinfI (Haemophilus influenzae strain Rf). on the other hand. restriction endonucleases with degenerate recognition sequences. and AluI (Arthrobacter luteus) cuts at AGCT. for instance. . so cuts at GAATC. meaning that they cut DNA at any one of a family of related sites. GAGTC. GATTC. A particular enzyme cleaves DNA at the recognition sequence and nowhere else. each distinguished by a slightly different mode of action. Type II restriction endonucleases. Sau3A (from Staphylococcus aureus strain 3A) recognizes GATC. five. Type II restriction endonucleases cut DNA at specific nucleotide sequences The central feature of type II restriction endonucleases is that each enzyme has a specific recognition sequence at which it cuts a DNA molecule. are the cutting enzymes that are so important in gene cloning. recognizes GANTC. and GACTC. PvuI (isolated from Proteus vulgaris) cuts DNA only at the hexanucleotide CGATCG. eight. or even longer nucleotide sequences.Three different classes of restriction endonuclease are recognized.

PvuII and AluI are examples of blunt end cutters. . resulting in a blunt end or flush end.almost all recognition sequences are palindromes: when both strands are considered they read the same in each direction. for example Blunt ends and sticky ends restriction endonucleases make a simple double-stranded cut in the middle of the recognition sequence.

so that the resulting DNA fragments have short single-stranded overhangs at each end These are called sticky or cohesive ends. Instead the cleavage is staggered. usually by two or four nucleotides. as base pairing between them can stick the DNA molecule back together again .Other Restriction Endonucleases cut double stranded DNA not exactly at same position as above.

which recognizes only the tetranucleotide GATC. The same sticky end is also produced by Sau3A.One important feature of sticky end enzymes is that restriction endonucleases with different recognition sequences may produce the same sticky ends. . BamHI (recognition sequence GGATCC) and BglII (AGATCT) are examples—both produce GATC sticky ends .

. GATC) should occur once every 44 = 256 nucleotides.e.g. These calculations assume that the nucleotides are ordered in a random fashion and that the four different nucleotides are present in equal proportions (i. and a hexanucleotide (e.The frequency of recognition sequences in a DNA molecule A tetranucleotide sequencen (e. . the GC content = 50%). GGATCC) once every 46 = 4096 nucleotides...g.